Precursor and effector CTL specific for determinants present on allogeneic MHC class I molecules have previously been shown to bear the Lyt2 cell surface phenotypic marker. Lyt2 is believed to be involved in the function of these cells because antibodies directed against Lyt2 block the cytolytic activity of most in vitro-generated MHC class I allospecific mature CTL. Based on those studies, Lyt2 has been proposed to be either an accessory molecule contributing to antigen specificity, or an "off" signal in mature. CTL. Our studies have focused on the role of Lyt2 during the initial activation of class I-specific CTL in vitro. CTL induced in vitro in the presence of anti-Lyt2 monoclonal antibody are resistant to subsequent blocking of lytic effector function by anti-Ly2 antibody, in sharp contrast to CTL generated in conventional (antibody-free) cultures. We have used limiting dilution assays to demonstrate that anti-Lyt2 monoclonal antibody actively induces the generation of Lyt2-resistant CTL effectors cells, and does not merely permit the selective outgrowth of a minority population of Lyt2-resistant precursor CTL. Furthermore, in contrast to conventional class I-specific effector cells, CTL activated in the presence of anti-Lyt2 antibody undergo down-modulation of their cell surface Lyt2, and exhibit an Lyt2-"dull" phenotype. We are currently investigating the functional relationship between antibody-induced down- modulation of Lyt2 and Lyt2-resistant CTL activity. Since CTL with these unusual characteristics are generated only in the presence of anti-Lyt2 antibody, Lyt2 engagement by antibody during activation may initiate an alternative differentiation program in at least some precursor CTL. Thus, Lyt2 may function to generate an "on" signal during CTL activation.